Project Summary Toxoplasma gondii is a unicellular, obligate intracellular parasite that can infect almost any mammal or bird. Once inside the host organism it can invade and replicate within any nucleated cell and access privileged spaces such as the central nervous system. It is estimated that T. gondii infects 30% of humanity. Acute T. gondii infection (Toxoplasmosis) is characterized by uncontrolled proliferation of the parasite (the lytic cycle) where it repeatedly invades and replicates within host cells and then searches for new host cells to invade. Calcium signaling initiates the major events of the lytic cycle: motility, invasion, and egress. Oscillatory calcium changes in the parasite's cytosol allows for elegant responses in highly complex, and sometimes diverging pathways. While much is known regarding the downstream events that occur as a result of this specific calcium signaling, it is unknown how this parasite, and the many parasites it is related to, regulates calcium fluctuations at a molecular level, to induce highly specific responses. EF-hand containing calcium-binding proteins (CBPs) are a major mechanism used for controlling the intracellular free calcium concentration in eukaryotic cells. We hypothesized that a subset of highly expressed CBPs acts as key regulators of the diverging pathways that sustain the lytic cycle in T. gondii. We will determine the spatial and temporal patterns of transcript and protein expression in order to determine the regulatory network by which they drive and sustain invasion-linked traits (e.g., motility, invasion, and host cell egress). Expression patterns will be measured using qRT-PCR (transcript abundance). T. gondii-specific antibodies will be made and used for the quantification of protein expression using Western blotting. Spatial expression patterns of protein abundance will be conducted using immuno- fluorescent microscopy. The expression levels of CBPs will be manipulated both by constitutive overexpression or knock out of the genes in tachyzoites. CBP genes will be over-expressed using T. gondii-specific over- expression vectors. Knockout mutants for the CBPs will be obtained by homologous recombination using newly designed CRISPR/Cas9 gene-editing vectors that have been optimized specifically for T. gondii. Phenotypic characterization of mutants will include analysis of intracellular calcium regulation using newly designed parasites that constitutively express a fluorescent intracellular calcium probe (Genetically Encoded Calcium Indicator, or GECI-parasites). In total, this research will fully evaluate the spatial and temporal regulation of calcium by CBPs throughout the lytic stage, making subsequent strategies of parasite disruption through targeting of CBPs a compelling and realistic strategy for treating T. gondii infections.